Cloning Of The Napin Gene Promoter And Preliminary QTL Of Some Agronomical Import Traits In Brassica Napus

Rapeseed is one of the most important oil crops. It is planted all over the worldexcept in a few areas of Africa. Increasing rapeseed economical value needs theimprovement of plant variety of rapeseed at first, while the improvement is embodiedin the two aspects of quality and yield of rapeseeds. In the aspect of qualityimprovement of rapeseeds, one of the effective ways is the application of geneticengineering in the change of chemical composition and its content. And one of keytechnology is to regulate the expression of the excellent gene. The gene regulation ofthe higher plant mainly happens in the transcription level and is affected by theinteraction of various cis-acting and trans-acting factors. The gene promoter is animportant cis-acting factor in plant, a DNA sequence which is identified andcombined by RNA polymerase and thus initiating the gene transcription. Therefore,the cloning and validating of organic specific promoter in rapeseed is an importanttask.In the improvement of the rapeseed yield, it is of positive and practicalsignificance to make research on these quantitative trait loci (QTL) in the position ofthe rapeseed genome and its extent of influence on the phenotype, because therapeseed yield and the many other yield-related agronomic traits are manifested asminor genes controlled quantitative inheritance. In recent year, along with thedevelopment of quantitative genetics and modem biotechnology, the complexquantitative traits could be separated to many independent Mendelian factors by DNAmolecular marker and QTL mapping and determine its position in genome and itseffect. Meanwhile, the application of techniques such as MAS, transgene, etc. makesit possible to further improve the rapeseeds.1. Cloning of the promoter of gene Napin in Brassica napus Napin,a variety of seed storage protein encoded by multi-gene, exists widely inseed of Brassica species, occupying 20%～30% of total protein in seed. In the affectof ABA, napin gene is tissue-specifically expressed.A pair of primers was designed according to 5'non-encoding region of the napAsequence reported in GenBank. With as the genomic DNA of Brassica napus"Zhongshuang 4 hao" as the template, the amplified products by PCR was cloned intopMD 18-T Vector and sequenced. The results indicated the cloned fragment contained1159bp. Through analysis, the fragment had only a few nucleotides differences withthe reported napA, and had high homology with the other reported napin genepromoter, too.The fragment is rich in AT nucleotides, with the sequence features of higher plant,such as TATA-box, CAAT-box, G-box, and GATA-box. Furthermore, to investigatethe tissue-specific expression function of the napin gene promoter, the fragment wasinserted into pCAMBIA-1301 vector, in place of the promoter CaMV35S in the upperstream of GUS in pCAMBIA-1301 vector, and the GUS gene expressing vector ofpC1301.N was constructed. The recombined vector was transformed intoagrobacterium LBA4404. The Brassica napus seeds via ABA inducement wereinfected by pC 1301.N/LB4404, and then were dyed by GUS. The result proved thatthe napin gene promoter was seed specifically expessed.2. Preliminery QTL of some Agronomically Import Traits in Brassica napusRIL population was constructed using the F₁ descendants of combinationzhongshuang No.4×H228 constructed via seventh inbred. The 11 agronomic traits,such as plant height, No. of effective 1st branches, yield per plant, etc. wereinvestigated from parent and random 142 F₈ RIL population plant series. Thecorrelation analysis of the 11 traits in RIL population showed that plant height, No. ofeffective 1st branches, length of main inflorenscence, effective siliques of maininflorenscence, density of main inflorenscence, length of silique, seed per sillique,1000 seed weight, total effective siliques per plant had significant positive correlationwith yield per plant.Polymorphisms between zhongshuang No.4 and H228 was examined by SSR primers. 152 primers Showed polymorphisms, and a linkage map containing 123markers, including 18 linkage groups was constructed. The total genetic distance ofthe map was 3483.1cM, and the average distance of two adjacent markers was28.32cM. The distribution of marker in 18 linkage groups was not even. Meanwhile,the first, twelfth, thirteenth, seventeenth linkage groups contained more marks, whilethe rest had fewer.According to the analysis of traits mean variance and SSR discrepancy situ inRIL, the constructing of genetic map and QTL could be processed preliminary. But,the average of traits were not homogeneous in some sort, the RIL need was purifiedunceasingly. The genetic map and QTL in this paper had error in some sort. Thecompare of genetic map between Lowe's and ours showed 26 homology marker situ,which occupied 21.1% of the marker situ in the experiment.81 QTLs were detected for 11 agronomic traits. 4 QTLs were detected for plantheight, which explained 10.3%～28.9% of trait variance; 2 QTLs were detected forNo. of effective 1-st branches, which explained 22.1%～47% of trait variance; 16QTLs were detected for effective branches height, which explained 12.2%～51.8% oftrait variance; 15 QTLs were detected for length of main inflorenscence, whichexplained 7.4%～26.6% of trait variance; 5 QTLs were detected for effective siliquesof main inflorenscence, which explained 11.2%～25% of trait variance; 1 QTLs weredetected for density of main infiorenscence, which explained 17.3% of trait variance;12 QTLs were detected for length of silique, which explained 24%～36.7% of traitvariance; 2 QTLs were detected for seed per sillique, which explained 9.6% and16.9% of trait variance; 2 QTLs were detected for 1000 seed weight, which explained26%～13.7% of trait variance; 11 QTLs were detected for Total effective siliques perplant, which explained 14.8%～47.2% of trait variance; 11 QTLs were detected forplant height, which explained 14.3%～32.8% of trait variance.On the other hand, in this paper we also discussed on the expression of GUS, theefficiency of promoter, the genetic distance and effective factor, precision of QTL,etc.